| Blunt snout bream(Megalobrama amblycephala),also known as Wuchang bream,is one of the most popular freshwater fish in China with an annual output of 797,000 tons in 2015.M.amblycephala has a weak tolerance to glucose,hence feeding a high-carbohydrate diet is not conducive for ideal growth and feed utilization of the fish.In previous studies,intolerance to high glucose was found in the Topmouth culte(Erythroculter ilishaeformis Bleeker),Crucian carp(Carassius auratus gibelio),Blunt snout bream(Megalobrama amblycephala)and Black carp(Mylopharyngodon piceus)including depressed activity of glucose metabolism enzymes,and hyperglycemia.Other findings included a rise in the expression of stress proteins,hepatocyte swelling and pathological changes caused by high content of carbohydrates in the diet,which resulted in a nutritional and metabolic syndrome.Studies on fish glucose metabolism regulation mainly focus on insulin and insulin receptor substrate secretion,glucose transporter number and glucose metabolic enzyme activity,but rarely on signal transduction regulation of glucose metabolism.Recently,with the development of high-throughput sequencing(HiSeq)technology and bioinformatics,the regulation effects of microRNAs(miRNAs)on insulin secretion,blood glucose control and glucose metabolism in mammals have been determined.This study selected blunt snout bream as the research object,to further illuminate the glucose and lipid metabolism signal transduction mechanism through miR-34a targeted to SIRT1 by miRNA interference technology in vivo,through miRNAs transcriptome and target genes regulatory network analysis in response to a high-carbohydrate diet by means of HiSeq and bioinformatics.It provides regulating targets and molecular regulation strategies for improving glucose utilization and nutritional metabolic disorder,provides biological targets and control strategies,makes up for lack of fish nutrition research,and strives to achieve the unification of the academic study significance and application value.1.Three miRNA libraries from the intestine,liver,brain of Blunt snout bream fed a normal starch diet(NSD)and three intestine,liver,brain miRNA libraries of Blunt snout bream fed a high starch diet(HSD),were constructed using HiSeq technology.The results showed that the expression profile changed between different tissues(liver,intestine and brain),which indicated miRNAs regulation had high specificity to tissues.By contrast,differentially expressed miRNAs(DEMs)in intestine,liver and brain were about the glucose metabolism pathways and biological regulation after being fed a HSD.In addition,it was noted that DEMs not only regulated glycolysis/gluconeogenesis and glucose metabolism,but were also involved in PPAR signaling pathway and insulin signaling pathway through GO and KEGG analysis.There were 124 DEMs expressed significantly up or down in the liver.Biological functional annotation showed that DEMs target genes of three tissues were significantly enriched in pathways of glycolysis/gluconeogenesis,glucose metabolism,phosphoenolpyruvate carboxykinase activation,the citric acid cycle,adipocytokine signaling and insulin signaling.MiR-34a,an important miRNA regulating glucose metabolism,was obtained from DEMs according to the information reported in the literature,which might play an important role in lipid metabolism,cell proliferation and apoptosis.2.By the technology of rapid-amplification of cDNA ends(RACE)and molecular cloning,the complete DNA sequence of SIRT1 and FoxO1 of blunt snout bream were obtained.On analysis of the coding sequence,SIRT1 had the closest relationship with that of Sinocyclocheilus anshuiensis,and FoxO1 was similar with grass carp(Ctenopharyngodon idella)at up to 99%,which indicated that the SIRT1 and FoxO1 gene coding region were strongly conserved in the evolution of fish.Through expression analysis of histological localization,it showed tissue specificity of SIRT1 and FoxO1 gene and expression sensitive in kidney,liver,intestine and brain.3.Analysis and screening of differential expression genes(DEGs)and regulation pathways after silencing miR-34a in liver of blunt snout bream using the HiSeq technology,83265 independent genes and 25433 functional annotation genes were found.Compared with the control group,there was a total of 2212 DEGs,1183 of which were up-regulated in expression and 1029 were down-regulated.According to the KEGG database analysis,the most annotated genes were related to immune signaling pathways.It indicated miR-34a interference will affect the immune function of blunt snout bream,besides high glucose metabolism,which has laid a good foundation for nutrient metabolism and immune regulation studies on miR-34a of Megalobrama amblycephala.4.Aim to assess the effect on growth performance,lipid deposition and metabolism of blunt snout bream feeding on different carbohydrate level diets,using wheat starch as carbohydrate source and soybean oil as lipid source,two iso-nitrogenous semi-purified diets were formulated,including control group(nomal wheat starch level diet,NWS,25.85%digestible glucose,32.73%crude protein,8.54%crude fat)and high glucose group(high wheat starch level diet,HWS,45.25%digestible glucose,33.32%crude protein,8.54%crude fat).It was found that excessive dietary carbohydrate led to a nutritional metabolic disorder in blunt snout bream,reduced growth performance,and altered muscle fat content and the fatty acid composition.Excessive dietary carbohydrate intake also increased blood lipid levels and increased lipid metabolism enzymes activity in the liver,causing storage and vacuolar degeneration of lipid droplets in the liver.These results indirectly proved the consequent negative impact on the liver health of blunt snout bream and cause fatty liver disease.5.Feeding high carbohydrate diet for 8 weeks,SIRT1 gene expression of blunt snout bream depressed,promoting deacetylation of FoxO1 and mRNA expression.Under regulation of SIRT1/FoxOl pathway,PEPCK and G6PD expression was activitied,promoting gluconeogenesis,and PPAR? expression promoted fatty acid synthase and lipid metabolism enzymes up regulation,such as FAS,lipase and ACC,leading to transfer excessive glycogen into lipid deposition in liver.In addition,the expression of inflammatory factor TNF? and tumor protein p53 were up-regulated,which indicated that high glucose caused the inflammatory reaction and apoptosis of cells.6.Two models of High glucose metabolism inhibited and normal carbohydrate metabolism activated have been constructed through miR-34a silencing and overexpression.Protein and gene mRNA expression of SIRT1,FoxO1,glucose metabolism enzyme,lipase enzyme and anti-inflammatory factor,miR-34a transcriptional was depressed because of miR-34a silencing in blunt snout bream fed a high wheat starch diet,which targeted SIRT1 and promoted SIRT1 protein expression by inducing deacetylation.SIRT1 activated transcriptional expression of FoxO1 by deacetylation,and changed from nucleus into cytoplasm,which affected the insulin signaling pathway,depressed hepatic gluconeogenesis,reduced liver glycogen accumulation and accelerated lipid decomposition and fatty acid synthesis.Meanwhile,the over expression of SIRT1 caused expression depression of TNFa and p53,which resulted in an inflammatory reaction and hepatic cell apoptosis decreased.Overexpression of MiR-34a decreased SIRT1 deacetylation and protein expression in the liver under physiological status of glucose homeostasis.It up-regulated FoxO1 and p53 mRNA expression,and accelerated the decomposition of fat into fatty acids.In this case,the synthesis of lipids was promoted and gluconeogenesis accelerated,resulted in inhibition of SIRT1 protein expression.These results also indicate that,in states of high glucose and glucose homeostasis,miR-34a regulates glycolipid metabolism and immunity performance as a reversible regulation mechanism targeting SIRT1 as a core factor. |
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